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Cell Reports MethodsBiochemistryPeer-reviewed journal article

Charting the molecular landscape of neuronal organisation within the hippocampus using cryo electron tomography

Calina A. GlynniD, Jake L. R. SmithiD, Matthew Julian Case, Rebecca Csöndör, Ana Katsini, Maria E Sanita, Thomas S. GleniD, Avery PenningtoniD, Michael GrangeiD

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Abstract

Cellular cryo-electron tomography (cryoET) enables the capture of detailed structural information within a biologically relevant environment. However, information in more complex samples, such as tissues, is lacking. Importantly, these observations need to be set in context of populations; imaging on the molecular scale to-date is limited to few observations in-situ that struggle to be generalised. This is due to limitations in throughput and versatility employed by current instrumentation. Here, we utilise plasma focused ion beam milling to examine the molecular landscape of mouse hippocampus by cryoET in targeted regions across multiple individuals revealing the complex organisation of macromolecules from the CA1 strata pyramidale (sp) to radiatum (sr). Our data represent a molecular atlas, producing snapshots of hippocampal architecture in adult mouse. The combination of instrumentation and application of technical advancements provides a framework to explore specific structural questions within native tissues in a targeted manner.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (16 Oct 2024) · CC BY 4.0

Xenon plasma FIB cryo lift-out from high-pressure-frozen mouse hippocampal slices up to 200 µm thick enables fluorescence-guided cryoET of CA1 synapses and apical dendrite cytoskeleton.

Tissue biopsies are roughly 1000-fold thicker than the 200-300 nm sections cryoET requires, and gallium FIB lift-out reaches only 30–50 µm depths while on-grid freezing compresses tissue. Xenon plasma FIB milling of uncompressed high-pressure-frozen slices enables deeper, layer-targeted molecular imaging repeatable across individual mice.

Key results

  1. ShownXenon plasma FIB trenching and undercuts let serial lift-out extract and deposit sections from thick high-pressure-freezing (HPF) carriers with high retention, and hybrid xenon-then-argon thinning produced lamellae of thickness comparable to cellular lamellae.

    26 lift-outs gave 186 serial sections, of which 170 (91.4%) survived transfer between microscopes; a ∼60-70 µm perpendicular lift-out yields 10-14 sections; xenon mills 3-4 times faster than argon; thinned lamellae had contrast transfer functions fit to sub-nanometre resolution. · Extraction of brain tissue from high pressure…

  2. ShownScreening tissue thickness, cryoprotectant and incubation identified formulations that vitrify uncompressed brain slices in HPF carriers, including when cryoprotectants are mixed into maintenance buffers such as artificial cerebrospinal fluid.

    Of 18 conditions, the most robust combined at least 15 min incubation with at least 10% low-molecular-weight cryoprotectant (sucrose 0.34 kDa or ethylene glycol 0.062 kDa) plus dextran (40 kDa); dextran- or BSA (66.5 kDa)-only mixes failed, giving distorted membranes or ice reflections in tilt images. · Brain tissue vitrification; Discussion

  3. ShownFluorescence-guided lift-outs placed 50-150 µm below CA1 stratum pyramidale (sp) nuclei sampled process-rich stratum radiatum (sr) neuropil with few ribosomes, unlike ribosome-dense CA1-sp tomograms, consistent with expected layer composition.

    Six datasets from 2 mice, 359 tilt series: vesicle-containing processes 311/359 (87%), microtubules 302/359 (84%), mitochondria without granules 119/359 (33%), actin 114/359 (32%), small ribosome clusters 60/359 (17%), synapses 56/359 (3-30% of tomograms per dataset). · Hippocampal layer targeting using cryo-CLEM

  4. ShownVitrified CA1-sr synapses preserved cleft-spanning densities, vesicle tethers and rare vesicle fusion intermediates; vesicle size matched prior cryo-preserved values and exceeded that of resin-embedded synapses.

    Mean synaptic vesicle diameter 45.4 ± 2.2 nm (per-tomogram averages 41.2-51.2 nm), with no relationship to mouse sex, cryoprotectant/buffer, post-mortem interval or collection day; fusion events in 8 of 107 synapses; cleft membranes ∼20-30 nm apart. · Synapse Organization within the CA1-sr; Discussion; Tomogram Segmentation

  5. ShownPlanar lift-out spanning CA1 strata oriens to lacunosum moleculare produced spatially ordered lamellae with axial views of apical dendrites, resolving 13 and 14 protofilament microtubules, lumenal particles, microtubule-ER tethers and putative inter-microtubule bridging tethers.

    Lift-outs of 350 and 390 µm yielded 40 and 42 sections; 13 imaged lamellae from one gave 246 tomograms over 150 µm below CA1-sp, with axial dendrite views in 25/246 vs 1/359 for perpendicular lift-outs. In 26 dendrite tomograms, microtubule nearest-neighbour spacing averaged 57.7 nm (median 54.0 nm, n=481), with no significant change by distance. · Assessment of the molecular organisation of…; Analysis of the apical dendrite cytoskeleton…

Methods, data and reproducibility

Acute mouse brain slices (100-200 µm) were Hoechst-stained, cryoprotected and high-pressure frozen in carriers; cryo-confocal and in-chamber fluorescence maps guided xenon plasma FIB serial lift-out in perpendicular or planar geometry, then xenon/argon thinning, 300 kV tilt-series cryoET, and segmentation-based quantification.

  • Serial lift-outs (perpendicular and planar) from high-pressure-frozen brain slices · mouse (C57BL/6J) · n = 26 lift-outs; 186 sections (170 retained)
  • CA1 stratum radiatum perpendicular lift-out tilt series from 4–6-month-old male and female mice · mouse (C57BL/6J) · n = 359 tilt series; six datasets; 2 mice · EMPIAR-XXXXX (placeholder in text)
  • CA1 stratum pyramidale lift-outs targeting Hoechst-stained neuronal nuclei · mouse (C57BL/6J)
  • Planar lift-out spanning CA1 stratum oriens to lacunosum moleculare, one lift-out thinned and imaged · mouse (C57BL/6J) · n = 246 tomograms from 13 imaged lamellae (39 thinned)
  • Axial apical dendrite tomograms used for microtubule and cytoskeleton analysis · mouse (C57BL/6J) · n = 26 tomograms; 481 microtubules
  • Synaptic vesicle segmentation training set (tomograms with binary masks from Napari annotation and pseudo-labelling) · mouse (C57BL/6J) · n = 22 tomograms
  • Vitrification screen varying slice thickness, cryoprotectant, buffer and incubation, using mice aged 7 to 184 days · mouse (C57BL/6J) · n = 18 conditions
  • Cortex biopsy punch dataset from a 5-month-old female · mouse (C57BL/6J) · n = one mouse
Methods & tools
Helios G5 Hydra CX xenon plasma FIB/SEM (Thermo Fisher Scientific), Arctis plasma FIB with AutoTEM automated thinning, Meteor integrated fluorescence module (Delmic) with Odemis, Stellaris 8 Cryo Confocal Microscope with LAS X (Leica), Titan Krios G4, Selectris energy filter, Falcon 4i, Tomo5, Leica VT 1200S vibratome and high-pressure freezing, Warp (CTF estimation, motion correction), AreTomo (tilt-series alignment and reconstruction), IMOD, IsoNet (CTF deconvolution, missing-wedge correction), membrain-seg, Chimera Segger, Amira, Napari (segmentation), Volume Segmantics U-Net with ResNet50 encoder
Reagents & environment
Tomo5 5.17.0.6390; Warp 1.0.9; AreTomo 1.3.4; IMOD 4.12.56; Amira 2023.1.1; IsoNet; membrain-seg; Volume Segmantics U-Net (ResNet50 encoder, 13 epochs); custom NumPy/SciPy/scikit-image scripts.
Reproducibility gaps
EMPIAR accession is an unassigned placeholder (EMPIAR-XXXXX); no release location is given for the custom Python scripts (vesicle metrics, filament analysis) or the trained vesicle segmentation model.
Limitations (5)
  • Slices were held at 4°C for post-mortem intervals of 1.5 to 4 hours before freezing; the authors say HPF and vibratome steps must improve before near-pristine preservation from dissection to vitrification is achieved. · stated by the authors
  • Synapses are larger than the ∼300 nm electron mean free path, so a single tomogram cannot capture an entire synapse. · stated by the authors
  • The missing wedge clips vesicle tops and bottoms, so convex-hull volumes come out smaller and computed sphericity lower than in reality. · stated by the authors
  • The CA1-sr census rests on 2 mice and the dendrite cytoskeleton analysis on a single planar lift-out, so null results (vesicle size vs sex or post-mortem interval; microtubule spacing vs depth) have limited statistical power. · noted in this summary
  • Molecular identities such as Munc13-1-like tethers, cleft adhesion molecules and microtubule bridging or ER linkers are assigned by visual inspection; no subtomogram averaging or molecular labelling confirms them. · noted in this summary

Read this if you need a practical plasma FIB serial lift-out workflow for cryoET of thick high-pressure-frozen tissue, with cryoprotectant recipes and fluorescence-guided targeting of hippocampal layers.

Citations
34
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top 1%

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